Isolation and culture of Bone marrow-derived mesenchymal stem cells using a modified plastic-adherent method

Objective To isolate and culture high-grade mesenchymal stem cells from rats bone marrow and determine its property of plastic-adherent and specific cell surface markers.Methods Mesenchymal stem cells were isolated from male S-D rats bone marrow weighed 190-220g and cultured by using modified plastic-adherent method.After the first passage,the second passage would be conducted as the overall alignment of the cells reached 70-80%.Then the morphology and performance of the cells were observed with phase-contrast microscopy and the specific cell surface markers was also determined by flow cytometry.Results After the first passage there were round cells and the morphology of the cells were uniform showing typically plastic-adherent property.The time for the first adherence of 70-80% was 7-9days,and first passage to the second one and the second to the third one were 7-8days and 5-6 days,then 3-5×106 MSCs could be obtained from each rat.The positive rats of CD29,CD60 and CD44were 99.6%,94.9%.and 68.1%.While the negative rates of CD34 and Cd45 were 98.6% and 99.9%.Conclusions Reforming plastic-adherent method is a convenient and effective procedure without the need of centrifuge and other manipulation and generally excluded the non-cell pollution that reduced the damage to the cells and maintained the integrity of the cells,finally to ensured the procurement of relatively high-purity MSCs from rats bone marrow.